Project Summary Heart transplant graft survival is suboptimal with an average half-life of ~10 years. Devising new strategies to prolong heart transplant survival in humans represents an unmet need in transplantation medicine. The knowledge that regulatory T cells (Treg) are important regulators of injurious alloreactive T cell responses has led to attempts to shift the balance of the alloimmune repertoire toward regulation. One approach, the autologous transfer of Treg, presents disadvantages such as the instability of the regulatory phenotype post- transfer. We have obtained new interesting data indicating that, in stark contradiction with its described pro- inflammatory physiological role in ischemia reperfusion injury in transplant, treatment with exogenous interferon-beta (IFN?), a type I interferon, prolongs cardiac allograft survival in mice. Graft survival prolongation was accompanied by an increase in the number of total Treg concomitant with a sharp decrease in the donor- specific effector T cells (Teff) in mice treated with IFN?, suggesting a previously-unknown action of exogenous IFN? on endogenous Treg. Given the potent antiviral properties of type I interferons, their effects have been widely studied the context of innate immunity during viral infection with a strong focus on dendritic cells. However, their direct effects on T cells, which we contend could be prevalent during systemic delivery of an exogenous type I interferon such as IFN?, remain less known. To investigate the cellular and molecular mechanisms through which exogenous IFN? increases Treg and decreases donor-specific Teff cells in response to an allogeneic stimulus, we employed a combined computational and experimental approach. We first generated mechanistic hypotheses using a mathematical model and then tested them experimentally. This led to the hypothesis that IFN? directly acts on nave CD4+ T cells to increase Treg induction and on Treg to increase their stability and proliferation, thereby increasing the Treg to donor-specific Teff ratio and prolonging graft survival. Mechanistically, we contend that IFN? binding to the type I interferon receptor on the surface of nave CD4+ and Treg leads to a pSTAT activation pattern that induces the transcription of genes that encode proteins involved in Foxp3 acetylation and enhances IL-2 pro- survival signals. Herein we will identify the cellular mechanisms through which exogenous IFN? prolongs graft survival (Aim 1), elucidate the molecular mechanisms through which IFN? increases Treg (Aim2), and determine the interactions between IL-6 effects on T cells and IFN?-mediated increase in Treg (Aim 3), which will explore the potential synergistic efficacy of IFN? with ?IL6-R treatment (Tocilizumab) currently being tested in the clinic. This project has implications both improving transplant graft survival and as a proof-of-principle of a new integrated computational-experimental approach for identifying better transplantation therapeutics. ! !